Enhancement of dendritic branching in cultured hippocampal neurons by 17β-estradiol is mediated by nitric oxide

Enhancement of dendritic branching in cultured hippocampal neurons by 17β-estradiol is mediated by nitric oxide

Int. J. Devl Neuroscience 21 (2003) 225–233 Enhancement of dendritic branching in cultured hippocampal neurons by 17␤-estradiol is mediated by nitric...

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Int. J. Devl Neuroscience 21 (2003) 225–233

Enhancement of dendritic branching in cultured hippocampal neurons by 17␤-estradiol is mediated by nitric oxide T. Audesirk∗ , L. Cabell, M. Kern, G. Audesirk Biology Department, University of Colorado at Denver, P.O. Box 173364, Denver, CO 80217-3364, USA Received 17 April 2002; received in revised form 13 February 2003; accepted 14 February 2003

Abstract Both 17␤-estradiol (E2) and nitric oxide (NO) are important in neuronal development, learning and memory, and age-related memory changes. There is growing evidence that a number of estrogen receptor-mediated effects of estradiol utilize nitric oxide as an intermediary. The role of estradiol in hippocampal neuronal differentiation and function has particular implications for learning and memory. Low levels of estradiol (10 nM) significantly increase dendritic branching in cultured embryonic rat hippocampal neurons (158% of control). This study investigates the hypothesis that the estrogen-stimulated increase in dendritic branching is mediated by nitric oxide. We found that nitric oxide donors also produce significantly increased dendritic branching S-nitroso-N-acetylpenicillamine (SNAP: 119%; 2,2 -(hydroxynitrosohydrazino)bis-ethanamine (NOC-18): 128% of control). We then determined that the increases in dendritic branching stimulated by estradiol or by a nitric oxide donor were both blocked by an inhibitor of guanylyl cyclase. Dendritic branching was also stimulated by a cell permeable analog of cyclic guanosine monophosphate (dibutyryl-cGMP: 173% of control). Estradiol-stimulated dendritic branching was reversed by the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide (carboxy-PTIO). This study provides evidence that estradiol influences the development of embryonic hippocampal neurons in culture by increasing the production of nitric oxide or by increasing the sensitivity of the neurons to nitric oxide. Nitric oxide in turn stimulates dendritic branching via activation of guanylyl cyclase. © 2003 ISDN. Published by Elsevier Science Ltd. All rights reserved. Keywords: Hippocampal neurons; 17␤-Estradiol; Nitric oxide

1. Introduction Both 17␤-estradiol (E2) and nitric oxide (NO) play critical roles in neuronal development, learning and memory, and age-related memory changes (for estradiol, see reviews by McEwen and Alves, 1999; Woolley, 1999a,b; Lustig et al., 1994; Beyer, 1999; for NO, see reviews by Haley, 1998; Hawkins et al., 1998; McCann, 1997; Meyer et al., 1998; Yun et al., 1996). Nitric oxide is generated by nitric oxide synthase (NOS), of which three isoforms have been identified: neuronal NOS (nNOS, also called NOS-I), endothelial NOS (eNOS, NOS-3) and inducible NOS (iNOS, NOS-II). There is growing evidence that E2 influences NO production through both genomic and non-genomic pathways. For example, estradiol enhances transcription of eNOS in human endothelial cells (Kleinert et al., 1998) osteoblast-like cells (Armour and Ralston, 1998) and in skeletal mus∗

Corresponding author. Tel.: +1-303-556-2593; fax: +1-303-556-4352. E-mail address: [email protected] (T. Audesirk).

cle (Weiner et al., 1994). In fetal lamb pulmonary artery endothelial cells, E2 enhances eNOS protein and mRNA levels (MacRitchie et al., 1997) but also activates existing eNOS via a non-genomic mechanism (Chen et al., 1999). Non-genomic activation of eNOS by E2 has also been reported in H441 human airway epithelial cells (Kirsch et al., 1999). Endothelial release of NO is important in the vasodilation which gives rise to the cardioprotective effects of E2, and produces circulatory changes during pregnancy (see reviews by Hutchinson et al., 1997; Weiner and Thompson, 1997). Estrogen replacement therapy in post-menopausal women is associated with an increase in plasma nitrate/nitrite levels which are indirect indicators of NO levels (Lopez-Jaramillo and Teran, 1999). Estradiol has also been implicated both in increasing NOS activity and in increasing NOS mRNA levels in brain tissue (eNOS and nNOS in cerebellum: Weiner et al., 1994; nNOS in cerebellum: Hayashi et al., 1994; nNOS in hypothalamus: Ceccatelli et al., 1996). Within the hippocampus, CA1 pyramidal neurons have been reported to exhibit both nNOS and eNOS immunoreactivity (nNOS: Wendland

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et al., 1994; Iwase et al., 1998; eNOS and nNOS: Doyle and Slater, 1997; eNOS: Dinerman et al., 1994). Pyramidal neurons predominate in our hippocampal cultures. We recently demonstrated that low levels (10 nM) of 17␤-estradiol caused a consistent increase in dendritic branching in cultured rat hippocampal neurons. This effect was blocked by the estrogen receptor (ER) antagonist ICI 182,780, and did not occur in response to 17␣-estradiol, suggesting that E2 influences differentiation via activation of ER (Audesirk et al., submitted for publication). Here we investigate the hypothesis that E2 stimulation of dendritic branching in cultured rat hippocampal neurons is mediated via stimulation of NO production, which in turn exerts its effects through the activation of guanylyl cyclase, leading to increases in cyclic guanosine monophosphate (cGMP). The concentration of estradiol chosen for this study (10 nM) is a compromise between physiological levels (which are in the low picomolar range in human female CSF), and the desire to produce maximum effects during a limited time in culture. In neuroblastoma cells transfected with ER, 10 nM E2 was reported to stimulate the greatest increase in neurite length as well as maximal activation of gene transcription (Patrone et al., 2000).

2. Experimental procedures 2.1. Neuronal isolation and cryopreservation Hippocampal neurons were isolated from E18 embryos obtained from timed-pregnant Sprague–Dawley rats and cryopreserved using methods slightly modified from Mattson and Kater (1988) as previously described (Audesirk et al., 1991). Cryopreservation reduces the use of animals and produces a continuously available supply of cultures that are comparable to newly harvested hippocampal cells in the relative proportions the three cell types (pyramidal-like neurons, stellate neurons, and glial cells) that predominate in E18 hippocampal cultures (Mattson and Kater, 1988). Our 3-day cell survival rates (78.6%; see Table 1) approach those

reported by Mattson and Kater (1988) for fresh cultures after 2 days of growth. 2.2. Culture techniques Hippocampal neurons were thawed rapidly by adding 37 ◦ C medium to the freezing vial, then agitating it in a 37 ◦ C water bath. Neurons were cultured in gridded, poly-d-lysine (molecular weight approximately 150,000) coated 35 mm plastic culture dishes containing 2 ml of culture medium, at a density of approximately 150,000 cells per dish. At this density, the cells were sufficiently sparse to allow detailed morphological measurements. Cells were plated and allowed to attach to the dishes at 37 ◦ C in a humidified, 5% CO2 atmosphere for approximately 4 h in a medium consisting of Phenol Red-free Eagle’s Minimum Essential Medium (MEM) buffered with 10 mM sodium bicarbonate (pH 7.3) and 25 mM HEPES, and supplemented with 2 mM l-glutamine, 0.1% glucose (0.2% total glucose), 1 mM sodium pyruvate, 4% antibiotic mixture (penicillin/streptomycin/amphotericin B; Gibco), and 10% fetal bovine serum (low IgG FBS; HyClone). After 3–4 h (during which time the cells attached to the culture dish), the medium was replaced with medium of the same composition except that it contained 2% FBS and 2% antibiotic mixture. Cells were cultured for 3 days. Because Phenol Red has been shown to have estrogenic activity at concentrations typically present in culture medium (Hubert et al., 1986), Phenol Red-free medium was used. 2.3. Assessment of survival After the initial 3–4 h incubation, attached living cells were counted to provide a baseline for future determination of survival. Living cells at 4 h were firmly attached with no obvious vacuolization of the cytoplasm. Some were round and phase bright, while others were flattened and often phase-dark, frequently with small neurites. 2.4. Morphometric analysis

Table 1 Characteristics of hippocampal control cultures Parameter Percentage survival Percentage of neurons meeting pyramidal-like criteria Percentage of astrocytes Mean axon length (␮M) Mean dendrite length (␮M) Mean dendrite number Mean axon branches Mean dendrite branches a

Mean ± S.E.M. 78.6 ± 1.28a 69.9 ± 1.21a 9.6 217.6 17.0 6.3 19.3 1.0

± ± ± ± ± ±

0.94b 3.68a 0.30a 0.16a 0.67a 0.02a

Values represent approximately 90 control cultures from experiments described in this paper. b Values represent 18 control cultures; astrocytes counted individually.

After 3 days of incubation, the following parameters were measured on living cells and compared across control and experimental conditions, using a digitizing tablet and morphometric software (Sigmascan; Jandel Scientific): (a) survival (percentage of cells alive at 4 h that remained alive after 3 days; astrocytes having apparently undergone cell division to form a cluster were counted as one cell at 3 days to prevent their proliferation from biasing the data); (b) mean axon length (an axon is defined as a process at least twice as long as any other process; the presence of an axon is used to define a pyramidal-like cell; Mattson and Kater, 1988). As shown in Table 1, the average length of the neurites we defined as axons was over 12 times the length

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of the average dendrite, making the distinction very obvious; (c) mean number of branches per axon; (d) mean number of dendrites on pyramidal-like cells; (e) mean dendrite length of pyramidal-like neurons (not including dendritic branches); and (f) mean number of branches per dendrite of pyramidal-like neurons. All pyramidal-like cells encountered in predetermined fields were measured until 10 neurons had been evaluated in each dish. Each culture dish was considered a single data point, and at least 12 dishes were analyzed for each concentration of test substance and control. The dishes were coded and measured by an observer who was unaware of their treatment. The mean values for parameters measured in all the control cultures used in this study are shown in Table 1. 2.5. Statistics Statistics were performed using one-way ANOVA on raw data. Experimental data were graphed as a percentage of the control data for each experiment to facilitate comparisons. Statistical significance is indicated when P ≤ 0.05. The mean values of parameters for the control cultures used in this study are shown in Table 1. 2.6. Materials Cells were cultured in medium containing one or more of the following chemicals in the concentrations specified: 10 nM 17␤-estradiol (Sigma), 0.1% dimethyl sulfoxide (DMSO, used as a solvent and a solvent control), 10 ␮M S-nitroso-N-acetylpenicillamine (SNAP; an NO donor with an approximate 5 h half-life of NO release; Molecular Probes), 50 ␮M 2,2 -(hydroxynitrosohydrazino)bis-ethanamine (NOC-18; an NO donor with an approximately 20 h half-life of NO release; Calbiochem), 200 nM 1H(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ; a selective inhibitor of NO-sensitive guanylyl cyclase; Fisher), 100 ␮M dibutyryl-cyclic guanosine monophosphate (dbcGMP; a cell-permeable analog of cGMP; Calbiochem), 10 ␮M 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide (carboxy-PTIO; an NO scavenger; Calbiochem), and 5 ␮M hemoglobin (bovine erythrocyte; Sigma) The following inhibitors of NOS were used: 500 ␮M ethyl-N-phenylisothiourea (Calbiochem), 1 mM NG -nitro-l-arginine (RBI), 500 ␮M 1400 W (Calbiochem), and 500 ␮M 7-nitroindazole (RBI). 3. Results 3.1. Characteristics of control cultures These data are summarized in Table 1. Photographs of our cultures from cryopreserved cells at 48 h showing pyramidal-like neurons have been published (Kern and Audesirk, 1995; Audesirk et al., 1997). We have previously

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verified the distinction between neuronal (neurite-bearing) cells and astrocytes by immunohistochemical techniques (unpublished observations). As shown in Table 1, our control cultures are approximately 90% neuronal cells, approximately 70% of which met the criterion for the pyramidal-like neurons that we evaluated in this study. The remaining 10% of the cells are astrocytes. Neurites that we identified as axons in pyramidal-like neurons averaged over 12 times the length of dendritic neurites. Our apparent survival rates are unlikely to be influenced by cell proliferation, since previous studies indicate that the vast majority of hippocampal neurons harvested from E18 embryos are post-mitotic (Banker and Cowan, 1977). Further, the few clusters of astrocytes resulting from proliferation were counted as a single cell for survival assessment at 3 days. 3.2. Estradiol increases dendritic branching We have previously demonstrated a robust and highly significant stimulatory effect of 10 nM E2 on dendritic branching. The E2-stimulated enhancement of dendritic branching was reversed by ICI 182,780, and was not mimicked by 17␣-estradiol, supporting the hypothesis that E2 is acting via ER (unpublished observations). The enhancement of dendritic branching by E2 was confirmed in the present study (Fig. 1). 3.3. NOS inhibitors gave inconsistent results To test the hypothesis that E2 enhances dendritic branching by increasing NO production, we cultured neurons in three different NOS inhibitors. These inhibitors had diverse and inconsistent effects on cultured neurons over a 3-day exposure. This prevented us from using this approach to test the hypothesis that control levels of dendritic branching would be reduced by blocking endogenous NO production, or that E2-stimulated enhancement of branching would be reversed by blocking NOS activity. We attribute this lack of consistency to non-specific effects which become evident upon prolonged exposure of cultured neurons to the inhibitors. To our knowledge, there have been no other reports of detailed morphometric analysis of neurons cultured for a period of days in NOS inhibitors. 3.4. NO donors enhanced dendritic branching We then compared the effects of E2 to those of NO donors. The NO donor SNAP (10 ␮M), added to newly plated neurons, caused a significant enhancement of dendritic branching (but no change in any other developmental parameter) after 3 days in culture. We then repeated the experiment with an alternate NO donor, NOC-18 (50 ␮M). As shown in Fig. 2, both NO donors increased dendritic branching (NOC-18 also reduced survival and reduced axon length).

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Fig. 1. Estradiol (E2; 10 nM) significantly enhanced dendrite branching. In this graph, the E2 data are combined from an independent E2 experiment and from the E2 data shown in Figs. 4 and 5 (n = 36 cultures). Control values: survival: 82.69 ± 1.98; mean axon length: 223.73 ± 5.38; mean dendrite length: 17.0 ± 0.40; axon branches: 21.71 ± 1.27; dendrite branches: 0.96 ± .03; dendrite number: 6.97 ± 0.24. In all figures, indicates significance at P ≤ 0.05.

3.5. NO donor effects were reversed by guanylyl cyclase inhibitor ODQ Many, but not all, of the physiological effects of NO are mediated by NO activation of guanylyl cyclase to produce

cGMP (reviewed by McDonald and Murad, 1996; Murad et al., 1993; Schmidt et al., 1993). To gain insight into the mechanism of NO effects on dendritic branching, we cultured neurons under the conditions described above in the presence of SNAP (10 ␮M) and SNAP combined with ODQ

Fig. 2. NO donors SNAP (10 ␮M; n = 14) and NOC-18 (50 ␮M; n = 15) enhanced dendritic branching; estradiol data from Fig. 1 are included for comparison (n = 36). Control values for SNAP: survival: 72.41 ± 2.82; mean axon length: 241.29 ± 12.99; mean dendrite length: 15.67 ± 0.44; axon branches: 13.65 ± 0.89; dendrite branches: 1.07 ± 0.07; dendrite number: 5.35 ± 0.21. Control values for NOC-18: survival: 83.22 ± 3.06; mean axon length: 204.81 ± 6.67; mean dendrite length: 19.64 ± 0.34; axon branches: 18.27 ± 0.89; dendrite branches: 1.14 ± 0.07; dendrite number: 5.80 ± 0.21. Control values for estradiol: survival: 82.69 ± 1.98; mean axon length: 223.73 ± 5.38; mean dendrite length: 17.0 ± 0.40; axon branches: 21.71 ± 1.27; dendrite branches: 0.96 ± .03; dendrite number: 6.97 ± 0.24.

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Fig. 3. The guanylyl cyclase inhibitor ODQ reversed the effects of the NO donor SNAP on neuronal differentiation (n = 15). Control values: survival: 68.49 ± 2.46; mean axon length: 215.88 ± 7.70; mean dendrite length: 13.37 ± 0.39; axon branches: 15.56 ± 0.76; dendrite branches: 0.95 ± .05; dendrite number: 4.61 ± 0.20.

(200 nM), a selective inhibitor of NO-sensitive guanylyl cyclase. We found that ODQ, which had no significant effects by itself, completely reversed the effects of SNAP, eliminating the enhancement of dendritic branching (Fig. 3).

above in the presence of E2 (10 nM) and E2 combined with ODQ (200 nM). The results demonstrated that inhibiting guanylyl cyclase also blocked the effects of E2 (Fig. 4).

3.6. E2 effects were reversed by ODQ

3.7. E2 effects were reversed by the NO scavenger carboxy-PTIO

To determine whether the effects of E2 are mediated via cGMP, we cultured neurons under the conditions described

To determine whether the effects of E2 are mediated by NO production, we cultured neurons under the conditions

Fig. 4. ODQ (200 nM) reversed the effects of estradiol (10 nM) on dendritic branching (n = 12). Control values: survival: 81.81 ± 4.19; mean axon length: 212.82 ± 6.25; mean dendrite length: 19.22 ± 0.53; axon branches: 30.13 ± 1.76; dendrite branches: 0.97 ± .04; dendrite number: 7.73 ± 0.36.

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Fig. 5. The NO scavenger carboxy-PTIO (10 ␮M) reversed the effects of estradiol (E2; 10 nM; n = 11), supporting the hypothesis that the E2-stimulated increase in dendritic branching is NO-mediated (n = 11). Control values: survival: 74.31 ± 1.64; mean axon length: 214.98 ± 10.42; mean dendrite length: 17.78 ± 0.57; axon branches: 20.78 ± 1.49; dendrite branches: 0.86 ± .04; dendrite number: 7.86 ± 0.28.

described above in the presence of E2 (10 nM) and E2 combined with the NO scavenger carboxy-PTIO (10 ␮M). The results demonstrated that scavenging NO blocked the effects of E2 (Fig. 5). Hemoglobin is also frequently used as an NO scavenger. In our cultures, 5 ␮M hemoglobin alone selectively stimulated dendritic branching, producing results similar to those obtained with estradiol alone (data not shown),

so we were unable to utilize hemoglobin to scavenge extracellular NO. 3.8. Dibutyryl-cGMP enhanced neurite branching The data described above support the hypothesis that the effects of E2 on dendritic branching are mediated by

Fig. 6. Dibutyryl-cGMP (100 ␮M) significantly increased dendritic branching comparable to estradiol, and increased axonal branching to a lesser degree (n = 12). Estradiol data from Fig. 1 are included for comparison (n = 36). Control values for dibutyryl-cGMP: survival: 78.18 ± 2.84; mean axon length: 190.21 ± 6.52; mean dendrite length: 19.69 ± 0.67; axon branches: 23.76 ± 0.58; dendrite branches: 0.90 ± 0.03; dendrite number: 7.71 ± 0.30. Control values for estradiol: survival: 82.69 ± 1.98; mean axon length: 223.73 ± 5.38; mean dendrite length: 17.0 ± 0.40; axon branches: 21.71 ± 1.27; dendrite branches: 0.96 ± .03; dendrite number: 6.97 ± 0.24.

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E2-stimulated increases in NO production, which in turn activates guanylyl cyclase to produce cGMP. We tested this hypothesis further by culturing neurons in cell-permeable dibutyryl-cGMP to determine whether this would mimic the effects both of E2 and of exogenously supplied NO. Dibutyryl-cGMP substantially increased dendritic branching, and increased axonal branching significantly but much less dramatically, without affecting other parameters of differentiation (Fig. 6). These results provide further support for the hypothesis that activation of guanylyl cyclase by NO is the mechanism of enhancement of dendritic branching.

4. Discussion In several other studies, E2 has been reported to exert generally stimulatory effects on neurite production in cultured neurons. For example, in cortical cultures, E2 increased neurite branching and neurite branch length (Brinton et al., 1997) and neurite elongation (Zhang et al., 2000). Dendritic branching was enhanced by E2 in rat amygdala neurons (Lorenzo et al., 1992), while midbrain neurons treated with E2 show increased neurite branching and total neurite length (Beyer and Karolczak, 2000). Estradiol enhanced neurite outgrowth in cultured hypothalamic neurons (Ferreira and Caceres, 1991), in rat PC12 cells transfected with the human estrogen receptor (Lustig et al., 1994) and in estrogen receptor-transfected neuroblastoma cells (Patrone et al., 2000). Blanco et al. (1990) found that E2 supplementation produced longer axons in hippocampal cultures, a finding which differs from the present study. Differences between our data and those of Blanco et al. may result from different culture conditions. In the study by Blanco et al., hippocampal neurons from E19 (versus E18) embryos were cultured for 5 days (versus 3 days) in medium containing 10% (versus 2%) FBS and exposed to 100 nM (versus 10 nM) E2. Dendritic branching was not measured in the Blanco et al. (1990) investigation. Several studies implicate nitric oxide in neuronal differentiation. Nitric oxide donors released onto hippocampal granule cell dendrites produced a selective increase in dendritic MAP2 mRNA in vivo (Johnston and Morris, 1994). Hindley et al. (1997) reported that mouse hippocampal cultures treated with NO donors exhibited longer, more branched neurites and a higher proportion of neurite-bearing cells than untreated cultures. These authors also provided evidence that NGF-stimulated neurite outgrowth in PC12 cultures was enhanced by NO activation of guanylyl cyclase and was accompanied by increased cGMP production. Phung et al. (1999) found that NGF required the presence of nNOS to stimulate PC12 differentiation, but that guanylate cyclase production was not required for neurite production. Taken together, these findings suggest that, in PC12 cells, NO influences differentiation via both cGMP-dependent and cGMP-independent pathways. Using nNOS knockout

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mice, Inglis et al. (1998) reported significantly reduced dendrite branching in spinal motor neurons. Our data provide strong evidence that E2 stimulates dendritic branching via NO, whose effects are mediated by cGMP via guanylyl cyclase activation, a common NO signal transduction system (reviewed by McDonald and Murad, 1996; Murad et al., 1993; Schmidt et al., 1993). As noted earlier, many ER-mediated effects of E2 occur through enhanced production of NO. However, to our knowledge, there have been no previous studies linking E2 stimulation of neuritogenesis to enhanced NO production. Our data suggest a specific pathway, activation of guanylyl cyclase and increased cGMP production, by which NO stimulates dendritic branching. The ability of a single application of the NO donor SNAP (half life: 5 h) to enhance dendritic branching suggests that this effect is triggered relatively soon after plating, probably within the first day in culture. Because cultured hippocampal neurons contain more than one cell type, it is possible that the effects we measured in pyramidal-like neurons were the result of NO production by a different cell type. The NO scavenger carboxy-PTIO is a polar molecule which will not immediately enter cells; its reversal of the effects of E2 could be considered to provide support for a source of NO exogenous to the pyramidal-like cells we measured. However, during a 3-day exposure, we cannot rule out that carboxy-PTIO may enter neurons by endocytosis or other means and scavenge intracellular as well as extracellular NO. In fact, reversal of the effects of NO using this scavenger has been cited as evidence of endogenous NO production in other systems (Angotzi et al., 2002; Janssen et al., 1998). The data derived from carboxy-PTIO provide additional support for the role of NO in the enhancement of dendritic branching by E2, but do not reveal the source of NO. For this reason, we attempted to eliminate the possibility of an exogenous NO source by using an alternate scavenger, hemoglobin, which proved unsuitable due to its stimulation of dendritic branching. Thus, we cannot eliminate the possibility that the source of NO is exogenous to the pyramidal-type neurons that we evaluated, although this seems unlikely due to the sparseness of the cultures and the lability of NO. An extensive literature implicates E2 in stimulating dendritic spine production, increasing the number of excitatory synapses and sensitivity to stimulation, and enhancing LTP in CA1 hippocampal pyramidal neurons (reviewed by Woolley, 1998; Foy, 2001). However, the functional significance of enhanced dendritic branching stimulated by E2 in embryonic hippocampal pyramidal neurons is an unexplored area.

Acknowledgements This research was funded by grant ES10163 from the National Institutes of Environmental Health Sciences, and by grant AG19648 from the National Institutes of Aging.

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